This study aimed at gaining insight into the mechanism of interactions between sorghum starch and mushroom polysaccharides (MPs) obtained from three cultivars Lentinula edodes (LEP), Auricularia auricula (AAP), and Tremella fuciformis polysaccharides (TFP) in relation to gelatinization, retrogradation and digestibility of starch, and microstructures before and after digestion. MPs decreased the degree of gelatinization, activation energy (retrogradation energy), and digestibility of sorghum starch, whilst they increased the particle size of digesta in a concentration dependent manner (0.15–0.6%, w/v). Confocal laser scanning microscopic (CLSM) images of starch suspensions, gels, and digesta illustrated that LEP was able to coat the sorghum starch granules through electrostatic interaction, which promoted starch retrogradation in gels after long-term storage. AAP and TFP were found to possess ability to suppress starch crystallization in sorghum starch gels, achieved through hydrophobic interaction with starch. TFP (glucuronoxylomannan) and most AAP particles were unable to coat sorghum starch granules, although a small number of AAP particles complexed with the starch through hydrogen bonding. The interaction between sorghum starch and MPs depended on the nature of MPs and their concentration. These findings would provide fundamentals for the utilization of MPs as glycaemic controller of starch and starch-rich foods. Display omitted •Mushroom polysaccharides declined the degree of starch gelatinization and digestion.•Shiitake β-glucan interacted with sorghum starch via electrostatic interaction.•Ear mushroom glucuronoxylomannan hydrophobically interacted with sorghum starch.•Shiitake β-glucan promoted starch crystallization after long-term storage at 4 °C.•Glucuronoxylomannan suppressed sorghum starch crystallization during cold storage.